定点突变提高海洋氧化节杆菌KQ11右旋糖酐酶的催化活性  

Site-directed mutagenesis enhances the activity of dextranase from Arthrobacter oxidans KQ11

作　　者：夏冰冰 马岱 叶子凡 杨静文 张洪斌 胡雪芹[1] XIA Bingbing;MA Dai;YE Zifan;YANG Jingwen;ZHANG Hongbin;HU Xueqin(School of Food and Biological Engineering,Hefei University of Technology,Hefei 230009,Anhui,China)

机构地区：[1]合肥工业大学食品与生物工程学院,安徽合肥230009

出　　处：《生物工程学报》2024年第9期3072-3082,共11页Chinese Journal of Biotechnology

基　　金：安徽省自然科学基金(2108085MC120)。

摘　　要：右旋糖酐酶是一种专一性水解α-1,6糖苷键的酶。为了提高海洋氧化节杆菌(Arthrobacter oxidans)KQ11来源的右旋糖酐酶的酶活,本研究采用了定点突变的方法对参与“隧道状结合位点”的氨基酸进行改造,并在此基础上对507位进行了饱和突变,获得了酶活和催化效率提高的突变酶A356G、S357W、W507Y、W507F。与野生株(wild type,WT)相比,突变体W507Y的比活力提高了3.00倍,kcat提高了3.62倍,Km下降了54%,催化效率kcat/Km提高了8.98倍。三维结构分析表明,氢键数目的增加及“隧道状结合位点”间的距离是影响酶活的重要因素。相比于WT突变体,W507Y与“隧道状结合位点”的另一侧氨基酸间的距离缩短,更易产生氢键作用力,加快了底物的水解和产物排出,使得酶活和催化效率大幅提高。Dextranase is an enzyme that specifically hydrolyzes theα-1,6 glucoside bond.In order to improve the activity of dextranase from Arthrobacter oxidans KQ11,site-directed mutagenesis was used to modify the amino acids involved in the“tunnel-like binding site”.A saturating mutation at position 507 was carried out on this basis.The mutant enzymes A356G,S357W,W507Y,and W507F with improved enzyme activities and catalytic efficiency were successfully obtained.Compared with wild type(WT),W507Y showed the specific activity increasing by 3.00 times,the kcat value increasing by 3.62 times,the Km value decreasing by 54%,and the catalytic efficiency(kcat/Km)increasing by 8.98 times.The three-dimensional structure analysis showed that the increase of the number of hydrogen bonds and the distance between“tunnel-like binding sites”were important factors affecting enzyme activity.Compared with WT,W507Y had a shortened distance from the residues on the other side of the“tunnel-like binding site”,which made it easier to generate hydrogen binding forces.Accordingly,the substrate hydrolysis and product efflux were accelerated,which dramatically increased the enzyme activity and catalytic efficiency.

关 键 词：右旋糖酐酶 定点突变 比活力 底物通道 

分 类 号：Q814[生物学—生物工程]